Factor VIIa is a serine protease that participates in blood coagulation by activating factor X and/or factor IX. Factor VIIa is produced from its precursor, factor VII (FVII), which is synthesized in the liver and secreted into the blood where it circulates as a single-chain glycoprotein (MW=50,000).
Factor VII can in vitro be converted into the two-chain form factor VIIa by factor Xa, factor XIIa, factor IXa or thrombin. Several coagulation enzymes are capable of activating FVII, a process which is greatly enhanced by the binding to tissue factor and negatively charged phospholipids A (Nemerson and Repke, 1985, Thromb. Res. 40:351-358 and Silverberg et al., 1977, J. Biol. Chem. 252:8481-8488). Single chain FVII is converted to the two chain form, when bound in sufficient density to charged surfaces, (Radcliffe and Nemerson, 1975, J. Biol. Chem. 250:388-395 and Bjoern and Thim, 1986, Res. Discl. 269: 564-565) and evidence for autoactivation has been presented (Pederson et al., 1989, Biochemistry 28:9331-9336).
In the presence of tissue factor and calcium ions, factor VIIa, in vivo, is believed to convert factor X (FX) to factor Xa (FXa) by limited proteolysis. The latter enzyme in turn converts prothrombin to thrombin in the presence of factor Va (FVa), calcium ions, and phospholipid. Factor VIIa will also convert factor IX (FIX) to factor IXa (FIXa) in the presence of tissue factor and calcium.
Factor VII can be purified from plasma and activated into factor VIIa by for example, methods described by Broze and Majerus, 1980, J. Biol. Chem. 255: 1242-1247 and Hedner and Kisiel, 1983, J. Clin. Invest. 71:1836-1841. Factor VIIa may also be produced by recombinant DNA technology by culturing in an appropriate medium mammalian cells transfected with a DNA-sequence encoding factor VII, isolating the protein produced and activating said protein to factor VIIa (see, for example, European patent application no. 86302855.1).
The cDNA coding for human factor VII has been characterized (Hagen et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83: 2412-2416). The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature factor VII that circulates in plasma is composed of 406 amino acid residues. The amino acid sequence analysis of the activated protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VIIa by the cleavage of a single peptide bond between arginine (152) and isoleucine (153). This results in the formation of a two-chained molecule consisting of a light chain (152 amino acid residues) and a heavy chain (254 amino acid residues) that are held together by one disulphide bond. The light chain contains a .gamma.-carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule.
Biosynthesis of functional coagulation factor VII requires a vitamin K dependent posttranslational .gamma.-carboxylation of 10 glutamic acid residues in the N-terminal part of the molecule (Hagen et al., 1986, Proc. Natl. Acad. Sc. U.S.A. 83:2412-2416). The Gla-domain is involved in the Ca.sup.2+ dependent binding of FVII to negatively charged phospholipids associated with cell surface bound tissue factor (Sakai et al., 1990, J. Biol Chem., 265:1890-1894). Binding strongly promotes the conversion of the FVII zymogen to FVIIa, and also the enzymatic activity of FVIIa towards its substrates, FIX and FX, is profoundly enhanced (Nemerson and Repke, 1985, Thromb. Res. 40:351-358 and Silverberg et al., 1977, J. Biol. Chem. 252:8481-848).
Factor VIIa may be used in treating patients who have developed inhibitors to factor VIII (Hedner and Kisiel, 1983, J. Clin. Invest. 71:1836-1841) and for the treatment of patients suffering from bleeding disorders such as platelet disorders including thrombocytopenia, von Willebrand's disease and others typically present in association with severe tissue damages (European patent application no. 86309197.1).
Factor VIIa has been found to be a protein susceptible to proteolytic cleavage giving rise to a number of degradation products without clotting activity. Factor VII contains 17 lysine (positions 18, 32, 38, 62, 85, 109, 137, 143, 148, 157, 161, 197, 199, 316, 337, 341, 389) and 24 arginine (positions 9, 15, 28, 36, 79, 110, 113, 144, 152, 202, 223, 224, 247, 266, 271, 277, 290, 304, 315, 353, 379, 392, 396, 402) residues that are all susceptible to proteolytic degradation. The proteolytic cleavage may occur at different steps of the recovery procedure and also during storage. Degradation products have been observed both for factor VIIa derived from plasma as well as for factor VIIa produced by recombinant DNA technology.
As the degradation products are inactive molecules, their occurrence in the factor VIIa preparation will lead to a lower specific activity of the final preparation. Furthermore, the amount and nature of the degradation products may vary from one production batch to another giving rise to preparations with a variable content of biologically active factor VIIa.
One such proteolytic enzyme, cathepsin G, is a serine protease with chymotrypsin-like activity from human granulocytes (neutrophils), involved in connective tissue degradation. When the neutrophils are activated by external stimuli, these active proteinases are secreted. Systemic activation and excessive release of cathepsin G occurs in various diseased states such as septicemia and leukemia. The abnormal hemostatic balance observed under these conditions may result from cathepsin G mediated cleavage of the Gla-domains of coagulation factors. The enzyme cleaves predominantly after aromatic residues, with a substantial preference for Phe (Tanaka et al., 1985, Biochemistry 24:2040-2047).
Cathepsin G cleaves human FX and protein C between Phe-40 and Trp-41 (Turkington, 1991, Haemostasis 21:111-116 and Turkington, 1991, Thromb. Res. 63:399-406). This sequence is also present in FVII. The homologous position in human protein C contains a His, in contrast to the other vitamin K dependent coagulation enzymes. Human FX has, adjacent to Tyr-44, a lysine residue, which might lower the affinity for the cathepsin G (Tanaka et al., 1985, Biochemistry 24:2040-2047).
Factor VIIa preparations containing inactive degradation products will as mentioned have a less specific activity as compared to preparations in which all or a major part of the protein material is active. Accordingly, higher and more frequent doses are necessary to obtain and sustain a therapeutic or prophylactic effect as compared to a preparation with higher specific activity.
Variable amounts of inactive degradation products and as a consequence, variable content of biologically active factor VIIa will furthermore make calculation of appropriate doses troublesome and difficult, if not in some circumstances impossible.
Finally, a content of non-physiological degradation products in the final preparation may trigger the immune system of the patient. Re-administration may then result in allergic reactions, which in severe cases may have a lethal course. Patients may also develop high titers of antibodies against factor VIIa rendering subsequent treatment difficult or ineffective. Accordingly, a factor VIIa preparation with less tendency to proteolytic degradation in vitro will be more satisfactory and potentially more useful in factor VIIa therapy.
Although the exact half-life of factor VIIa is unknown, preliminary results suggest that factor VIIa procoagulant activity is rapidly cleared from the bloodstream upon intravenous administration (Hedner and Kisiel, 1983, Clin. Invest. 71:1836-1841).
The treatment and the lives of the patients will be negatively influenced by the observed short in vivo half life of native factor VIIa. Relatively high doses and frequent administration will be necessary to reach and sustain the desired therapeutic or prophylactic effect. As a consequence, adequate dose regulation will be difficult to obtain and the need for frequent intravenous administrations will impose restrictions on the patients' way of living.
Therefore, a need exists in the art for factor VIIa preparations which are stable during production, purification and storage even at high concentrations, and which furthermore have a longer half life and slower clearance from the blood than the native or recombinant factor VIIa. The present invention fulfills this need by providing certain modified factor VII/VIIa.